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The Within-Subject Application of Diffusion Tensor MRI and CLARITY Reveals Brain Structural Changes in Nrxn2 Deletion Mice Eleftheria Pervolaraki1†, Adam L

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The Within-Subject Application of Diffusion Tensor MRI and CLARITY Reveals Brain Structural Changes in Nrxn2 Deletion Mice Eleftheria Pervolaraki1†, Adam L Pervolaraki et al. Molecular Autism (2019) 10:8 https://doi.org/10.1186/s13229-019-0261-9 RESEARCH Open Access The within-subject application of diffusion tensor MRI and CLARITY reveals brain structural changes in Nrxn2 deletion mice Eleftheria Pervolaraki1†, Adam L. Tyson2,3,4†, Francesca Pibiri5, Steven L. Poulter5, Amy C. Reichelt6, R. John Rodgers7, Steven J. Clapcote1, Colin Lever5, Laura C. Andreae2,3† and James Dachtler1,5*† Abstract Background: Of the many genetic mutations known to increase the risk of autism spectrum disorder, a large proportion cluster upon synaptic proteins. One such family of presynaptic proteins are the neurexins (NRXN), and recent genetic and mouse evidence has suggested a causative role for NRXN2 in generating altered social behaviours. Autism has been conceptualised as a disorder of atypical connectivity, yet how single-gene mutations affect such connectivity remains under-explored. To attempt to address this, we have developed a quantitative analysis of microstructure and structural connectivity leveraging diffusion tensor MRI (DTI) with high-resolution 3D imaging in optically cleared (CLARITY) brain tissue in the same mouse, applied here to the Nrxn2α knockout (KO) model. Methods: Fixed brains of Nrxn2α KO mice underwent DTI using 9.4 T MRI, and diffusion properties of socially relevant brain regions were quantified. The same tissue was then subjected to CLARITY to immunolabel axons and cell bodies, which were also quantified. Results: DTI revealed increases in fractional anisotropy in the amygdala (including the basolateral nuclei), the anterior cingulate cortex, the orbitofrontal cortex and the hippocampus. Axial diffusivity of the anterior cingulate cortex and orbitofrontal cortex was significantly increased in Nrxn2α KO mice, as were tracts between the amygdala and the orbitofrontal cortex. Using CLARITY, we find significantly altered axonal orientation in the amygdala, orbitofrontal cortex and the anterior cingulate cortex, which was unrelated to cell density. Conclusions: Our findings demonstrate that deleting a single neurexin gene (Nrxn2α) induces atypical structural connectivity within socially relevant brain regions. More generally, our combined within-subject DTI and CLARITY approach presents a new, more sensitive method of revealing hitherto undetectable differences in the autistic brain. Keywords: MRI, CLARITY, Social, Autism, Axons, Diffusion, Structure, Imaging * Correspondence: [email protected] †Eleftheria Pervolaraki, Adam L. Tyson, Laura C. Andreae and James Dachtler contributed equally to this work. 1School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, UK 5Department of Psychology, Durham University, South Road, Durham DH1 3LE, UK Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Pervolaraki et al. Molecular Autism (2019) 10:8 Page 2 of 13 Background Currently, it is unknown whether deletion of Nrxn2α Autism is a common neurodevelopmental disorder, which changes the brain’s microstructure and connectivity. One is highly heritable [1]. While heritability is high, it is also previous study found coarse alterations to cell layer thick- clear that autism is highly polygenic. Around ~ 400–1000 ness within the hippocampus of Nrxn2α homozygous genes are involved in autism susceptibility [2–5]. Many of KOs [20]. However, cell density measurements are un- these genes cluster upon proteins relating to synaptic sig- likely to reveal the true extent of changes within the autis- nalling [6]. A family of presynaptic proteins garnering re- tic brain. Within the current study, we have addressed this cent interest have been the neurexins (NRXNs). NRXNs by developing a dual imaging approach (DTI and CLAR- are encoded by three genes (NRXN1, NRXN2, NRXN3; ITY) that quantifies the alignment and density of white note that CNTNAP1 and CNTNAP2 are sometimes re- matter, applied here to brain regions known to support so- ferred to as NRXN4), of which two major isoforms exist: cial behaviour in a mouse model of autism. the longer α proteins with six laminin/neurexin/sex hor- Diffusion tensor MRI (or DTI) is based upon the mone (LNS) binding domains, and the shorter β proteins movement of water molecules, a measure that is termed with one LNS binding domain [7, 8]. fractional anisotropy (FA). Apparent diffusion coefficient Mutations within all three NRXN genes have been linked (ADC) is similar to FA but quantifies diffusion restric- to autism [6]. Heterozygous deletions within NRXN2 have tion as opposed to the spatial symmetry of diffusion. been identified in a number of individuals with autistic phe- This approach has been used to explore neuropatho- notypes. These include an autistic boy and his father (who logical markers in autistic patients; alterations in myelin- had severe language delay but not autism) who both had a ation, axonal abundance, size and orientation all modify frameshift mutation within exon 12 of NRXN2 [9]; a FA and ADC values [21–23]. Using the preferred direc- 570-kb de novo deletion of 24 genes at chromosome tion of the diffusion of tensors between brain regions 11q13.1, including NRXN2, in a 21-year-old man displaying can be used to explore their potential connection. Quan- a clinical phenotype including autistic traits [10]; a 1.6-Mb tification of those computed streamlines by FA and axial deletion at chromosome region 11q12.3–11q13.1, including and/or radial diffusion can indicate impairments in re- NRXN2, in a 23-year-old man with intellectual disability gional structural connectivity. Since aberrant brain con- and behavioural problems [11]; a de novo frameshift muta- nectivity is likely a core feature of autism [24], we tion identified in a Chinese man with autism spectrum dis- reasoned that the candidate method for probing the aut- order (ASD) [12], a 921-kb microdeletion at 11q13 in a istic brain should combine tractographic techniques. Ac- 2-year-old boy who had language and developmental delay cordingly, here, we combined high-resolution imaging of (although did not meet the autism diagnosis criteria) [13] labelled neuronal tracts in brains rendered transparent and a paternally inherited microRNA miR-873-5p variant by CLARITY with DTI. in an ASD individual which altered binding affinity for sev- CLARITY is a recent development that renders tissue eral risk genes including NRXN2 and CNTNAP2 (NRXN4) optically transparent and macromolecule permeable [14]. Furthermore, recently, two large-scale reports have [25]. This permits antibody staining and imaging of identified NRXN2 with ASD risk. A study of 529 ASD pa- much larger tissue volumes than possible under trad- tients and 1923 controls in a Chinese population identified itional immunofluorescence techniques. By examining two NRXN2 variants which significantly increase ASD risk fibre orientation without sectioning-related artefacts and [15]. The second study employed machine learning ap- biases, axonal staining in cleared tissue affords a deeper proaches across 5000 ASD families to rank the importance understanding of the microstructure and structural con- of ASD candidate genes and ranks NRXN2 in the top ~ nectivity of a brain region. 0.5% of genes, i.e. 113th [16]. For comparison, NRXN1,for Given the social impairments found within Nrxn2α which the evidence base for its links to ASD is broader and mice, we sought to examine those brain regions most stronger, ranks 45, and CNTNAP2 ranks 211th [16]. Con- closely linked with social behaviour (see Additional file 1: sistent with these association studies, we and others have Supplemental materials). Briefly, we identified four re- previously found that homozygous or heterozygous gions of interest (ROIs): the amygdala and three brain deletion of Nrxn2α induces impairment in social ap- regions strongly and directly connected to the amygdala; proach and social recognition [17–19]. In summary, the hippocampus, orbitofrontal cortex (OFC), and anter- although mutations within NRXN2 are rare, under- ior cingulate cortex (ACC). As predicted, structural con- standing how they may drive social, ASD-relevant be- nectivity was abnormal in Nrxn2α mice. havioural changes is important. One important goal is to help elucidate how apparently convergent patho- Methods physiology in ASD emerges despite marked genetic Ethics heterogeneity [5]; mapping brain alterations driven by All procedures were approved by the University of Leeds different single genes is thus a crucial task. and Durham University Animal Ethical and Welfare Pervolaraki et al. Molecular Autism (2019) 10:8 Page 3 of 13 Review Boards and were performed under UK Home Of- gradient vector on the x, y and z orientations. Unwanted fice Project and Personal Licenses in accordance with background, setting a threshold, smoothing of the data the Animals (Scientific Procedures) Act 1986. and definition of tissue boundaries were performed prior to the reconstruction of the final 3D image.
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